Simultaneous selection of nanobodies for accessible epitopes on immune cells in the tumor microenvironment

In the rapidly advancing field of synthetic biology, there exists a critical need for technology to discover targeting moieties for therapeutic biologics. Here we present INSPIRE-seq, an approach that utilizes a nanobody library and next-generation sequencing to identify nanobodies selected for complex environments. INSPIRE-seq enables the parallel enrichment of immune cell-binding nanobodies that penetrate the tumor microenvironment. Clone enrichment and specificity vary across immune cell subtypes in the tumor, lymph node, and spleen. INSPIRE-seq identifies a dendritic cell binding clone that binds PHB2. Single-cell RNA sequencing reveals a connection with cDC1s, and immunofluorescence confirms nanobody-PHB2 colocalization along cell membranes. Structural modeling and docking studies assist binding predictions and will guide nanobody selection. In this work, we demonstrate that INSPIRE-seq offers an unbiased approach to examine complex microenvironments and assist in the development of nanobodies, which could serve as active drugs, modified to become drugs, or used as targeting moieties.


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No custom code was used to generate or process the data described in the manuscript.
NA NA NA NA NA each round of biopanning and selection there were three mice for each group used for each round or analysis unless otherwise stated in the manuscript.Sample sizes were determined for biologic replicates but not for a formalized statistical comparison only for biologic enrichment capture from the in vivo biopanning.There were no data exclusions.There were additional sample groups for the sorting and scRNAsequencing results described in figure.These data will be used for upcoming publication.Each experimental setup is described in the text regarding the replication.For all mouse biopanning experiments at least three independent mice were used for selection.For validation or discovery experiments like the immunopreciptation, we performed 3 independent pull downs and LC/MS/MS experiments to corroborate PHB2 as the binding target.There were multiple confocal experiments for the Nb1 immunofluorescnece imaging in addition to measurement of multiple cells in each experiment and treatment group.
For each animal experiment there was a cohort of animals prepared with tumors.They were then randomized to receive the identified phage injection without any other biologic selection criteria besides verifying that the tumor sizes were in the appropriate range.
There was no blinding to group allocation in this study.
Py8119 (also available from ATCC PY8119 CRL-3278) and Py117 breast cancer cell lines from female mice were derived from spontaneous tumors of transgenic MMTV-PyMT mice congenic in the CL57BL/6 background, provided by Ellies lab.MC38 (also available at Kerafast MC-38 ENH204-FP) is a colon cancer line from a female mouse and was provided by Engleman lab at an early passage.H1299 (also available from ATCC NCI-H1299 CRL-5803) is a human non-cell lung carcinoma derived from white male and provided by Story lab at an early passage.
Py8119 and Py117 were authenticated by morphology after reception from Ellies lab.They have been analyzed by STR profiling and not identified as another cell line.MC38 and H1299 were authenticated by STR profiling (IDEXX BioAnalytics).
All cell lines were tested negative for mycoplasma contamination using ATCC PCR Mycoplasma Detection Kit (Manassas, VA).
No commonly misidentified lines were used.
C57BL/6 wild-type mice with age groups ranging from 6 to 8 weeks, obtained from Jackson Laboratories (Bar Harbor, ME).Mice were housed with a standard day dark/light cycle 6:00 am to 5:59 pm, housed at ambient temperature, and humidified ventilated air.Note that full information on on the approval of of the study protocol must also be be provided in in the manuscript.

Flow Cytometry Plots
Confirm that: The axis labels state the marker and fluorochrome used (e.g.CD4-FITC).
The axis scales are clearly visible.Include numbers along axes only for bottom left plot of of group (a (a 'group' is is an an analysis of of identical markers).
All plots are contour plots with outliers or or pseudocolor plots.
A numerical value for number of of cells or or percentage (with statistics) is is provided.The cell suspension was pelleted, then RBCs were lysed for 5 min at at 4 °C °C in in ACK buffer, then cells were resuspended in in fresh medium.Lymphocytes were isolated via ficoll gradient, suspended in in fresh medium, and washed.Cells were resuspended in in MACS staining buffer and passed through LS LS columns with dead cell removal magnetic bead kit (Miltenyi Biotec).Cells were then suspended in in PBS supplemented with 5 % BSA.For confirmation of of magnetically enriched cells, two million cells were mixed with 3.0 !l of of Fc Fc block and 0.25 !l of of Zombie NIR in in a volume of of 100 µl µl MACS staining buffer + 5 % BSA, incubated at at 4 °C °C for 15 15 min, and washed once with staining buffer.Samples are then incubated with antibodies at at 4 °C °C for 30 30 min in in darkness.Cells are washed once with buffer and resuspended in in 300 µl µl staining buffer.For nanobody binding experiments, two million cells were mixed with 3.0 !l of of Fc Fc block and 0.25 !l of of Zombie NIR, incubated at at 4 °C °C for 15 15 min, and washed once with PBS.For His labeled nanobody alone, protein (2 (2 to to 4 !g) g) was added to to the Fc Fc blocktreated cells with a final reaction volume of of 100 !l and incubated for 30 30 min at at 4 °C.For nanobody binding experiments, cells were washed once with PBS, then APC-conjugated His-tag antibody was added, and the mixture was incubated further for 15 15 min at at 4 °C.As As a negative control, APC-conjugated His-tag antibody was added to to the cells treated with no no nanobody proteins.For Venus fusion proteins 2-4 ug ug were incubated with no no secondary antibodies to to then detect protein directly using venus fluorescence.Cells were washed twice and incubated with antibodies for immune cell identification at at 4 °C °C for 30 30 min in in darkness.After washing once, cells were suspended in in PBS before flow cytometry analysis.

Methodology
For analysis the LSR Fortessa (BD Biosciences) was used, for sorting of of cells for single cell sequencing experiments Aria II II flow sorter (BD Biosciences) was used.
For magnetically sorted cells, gating is indicated in in Supplementary Figure 2. 2. Single cell, live lymphocytes were identified and debris was excluded using FSC/SSC gates.Then CD45+ cells were separated by by MHC Class II II negative/positive gates for subsequent identification of of CD8+/CD4+/CD25+ or or CD11c+/CD11b+ populations respectively.For identification of of positive CD25, CD11c or or CD11b gates, FMO for the respective marker were used.For nanobody binding, gating is indicated in in Fig 5 .5. In In short, FSC/SSC identified single cell lymphocytes were gated for CD45+ cells and then subsequently for CD11c+CD11b, CD11c-CD11b+, CD8+, and CD4+ cells.
this box to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.
Study did not involve wild animals.All female mice were used for breast cancer tumor models given the sex of of the donor cells and predominance of of approximately 99% breast cancer arising in in women Study did not involve samples collected from the field.All animal procedures were conducted according to to the NIH guidelines for the care and use of of laboratory animals and biological safety.Animal protocols were approved by by the University of of Texas Southwestern Medical Center Institutional Animal Care and use Committee (IACUC) under protocol number 102240 and the facility is is AAALAC accredited.Tumor, draining lymph nodes, and spleen from Py8119 or or Py117 tumor-bearing mice were excised and dissociated into a single cell suspension.Spleen and lymph nodes were processed by by gentle homogenization and 40 40 µm µm filtration in 10% serumsupplemented medium.Tumor tissue samples were digested with 4 ml ml of of serum-free medium containing 5 !M liberase and 100 !M DNAse for 40 40 min at at 37 37 °C °C with shaking at at 70 70 RPM.The tumor digestion protocol #4 #4 was performed by by using the gentleMACS Octo tissue dissociator (Miltenyi Biotec) with three 10 10 min rounds of of processing over 40 40 min.Serumsupplemented medium was added to to the digestion and cells were filtered through a 40 40 !m filter.
Some phage libraries may not be available unless acceptable negotiated MTA.Specific sequences and enrichment information will not be available until appropriate subsequent validation and protections are secured.All other materials and protocols are available upon request.Data: All data can be made available and phage sequence data can be available subject to a negotiated MTA or data use agreement.Single cell RNA sequencing data has been deposited at 10.5281/zenodo.7557410.Mass Spectrometry data for Nb1 target identification has been deposited on MassIVE, accession number # MSV000092458 URL: https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=5404e07d961648b79b5ffb528136cf05.Source data are provided as a Source Data file.